Although lens induction in vertebrate embryos was discovered almost a hundred years ago by Spemann and colleagues, the molecular mechanism underlying this process is still unknown. It has recently been shown that the the transcription factors six3 and Pax6 have lens inducing activity in vivo. Expression of Six3 in medaka fish can induce lenses in otic vesicle while our own work has shown that Pax6 can induce lenses in multiple locations in the frog Xenopus leavis. In a collaborative effort between the Hemmati-Brivanlou and Lang labs, we propose to unravel the molecular pathways leading to lens induction in vertebrates. We will take advantage of the powerful expression cloning approaches available in Xenopus laevis to identify candidate lens inducing factors. These candidates will then be tested for their activity in mammals using the genetic systems available in the mouse. In our preliminary results, we describe two important advances. (1) Xenopus expression cloning of a factor with lens inducing activity. This factor (designated D4E) is unique, in that no orthologue exists in the sequences databases. However, the domain structure of the full-length molecule (designated 4E) clearly indicates that it has a role in signal transduction. The lens-inducing cDNA D4E encodes an N-terminaly truncated protein that is likely to act as a dominant negative mutant of full length 4E. (2) Identification of a lens-specific enhancer derived from the Pax-6 gene. This region of DNA directs transgene expression to the presumptive lens ectoderm only starting at embryonic day 8.75. Armed with these reagents, we propose in five specific aims to further characterize the activity of our novel gene 4E. In Xenopus, we will determining in which signaling pathway it functions and inquire about a possible regulatory relationship between 4E and Pax6. In the mouse we will use the lens-specific enhancer element to target 4E expression to the presumptive lens to ask about the function of 4E in-vivo. Finally, we propose to eliminate 4E from the mouse genome to determine if 4E is necessary for lens induction in mammals in vivo. We strongly believe that the comparative approaches proposed in this grant will allow the molecular resolution of one of the oldest embryological problems. Elucidating the molecular basis of lens induction in vertebrates will inevitably have a direct consequence on the rational design of drugs for the treatment of human lens diseases.